Alleviation of corn rootworm damage with microbial seed treatments

ABSTRACT

Corn rootworms are potentially devastating pests in corn production, especially in the corn belt of the United States. The microbial tools developed provide abilities to endophytically colonize plant roots. These endophytic microbes can be applied as seed treatments or in numerous other ways that allow them to colonize plant roots where they provide systemic beneficial changes in the plant physiology. Treatment with Trichoderma organisms on seeds alleviated damage on roots, even though the number of corn rootworm larvae on roots were not reduced.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/479,074, filed Mar. 30, 2017; U.S. Provisional Application No. 62/479,080, filed Mar. 30, 2017; and U.S. Provisional Application No. 62/479,084, filed Mar. 30, 2017. The entire contents of these applications is hereby incorporated by reference in their entirety.

TECHNICAL FIELD

This disclosure relates to the control of corn rootworms using microbial seed treatments.

BACKGROUND ART

Corn rootworms (“CRW”) (Diabrotica spp.) are potentially devastating pests in corn production, especially in the corn belt of the United States. Damage exceeds $1 billion per year. Eggs of the pest are deposited in soils in the summer and over winter to hatch in late May or early June. They begin to feed on corn roots and go through three larval instars during their feeding on roots. After feeding for several weeks they molt and the adults winged versions emerge into corn fields. The adults (known as cucumber beetles) feed on corn pollen and silks and may damage silks in conditions of heavy infestation and prevent pollination. They also eat leaves of corn and other crops, and they may carry viruses that infect and damage cucumbers. At the end of the season, they lay eggs in soil that hatch as noted above to feed on corn roots. The primary and greatest damage by these insects is from feeding on corn roots.

It is also important to note that CRW damage to corn roots can offer windows of opportunity for infection by pathogenic fungi in soil. Further, nematode damage to roots can cause a significant increase in root damage and function. The combined effects of CRW, disease and nematodes all impact the function of roots. Damage to root function not only reduces yields under ideal conditions, but especially under stressful conditions (e.g. drought or soil compaction) the effects are even more devastating.

SUMMARY

The applicant is involved in research to provide products that improve plant performance, including SABREX and GRAPHEX. The former product is intended for use with corn (Strain K2 (T. afroharzianum)+Strain K4 (T. atroviride)), while the latter is an inoculant seed lubricant used with soybeans.

It is an ongoing concern in agriculture to provide enhanced root development and the work reported herein is a part of the effort to provide such development. These and other advantages are provided by the compositions and methods described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing root damage ratings of corn plants grown with several different microbial treatments.

FIG. 2 is a chart showing plant height of corn plants grown with several different microbial treatments.

FIG. 3 is a chart showing root weight of corn plants grown with several different microbial treatments.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

At the outset, it should be clearly understood that like reference numerals are intended to identify the same structural elements, portions or surfaces consistently throughout the several drawing figures, as such elements, portions or surfaces may be further described or explained by the entire written specification, of which this detailed description is an integral part. Unless otherwise indicated, the drawings are intended to be read together with the specification, and are to be considered a portion of the entire written description of this invention.

There are potentially several methods of control of damage of CRW larvae, none of which are more than partially effective. These follow, with a discussion of their limitations.

Chemical Control.

A number of insecticides are toxic to the larvae. However, seed treatment chemicals are effective only on or very near the seed. Since CRW eggs are generally distributed throughout the soil, most of the insects will encounter roots distant from the seeds, and so these chemical seed treatments are not very effective.

Prophylactic insecticide applications may be applied, but they are expensive and only give a positive return to investment to farmers about 15% of the time. Other materials such as carbofuran may be applied post-emergence, but timing is critical. If the material is applied too early, the product will leach and degrade before the CRW are present to control. Timed too late and the product may not have time to reach the root zone to control the pest before it damages the crop. This material should be explicitly timed to 1 week before to 1 week after 5% CRW egg hatch (entrance into the 1st instar).

In all cases, the decision to use an insecticide is an economic one and probably requires the use of scouting to determine the level and timing of infestation and development of the larvae. Thus, chemical control may be essential to reduce losses under severe levels of the pest, but it also has major limitations.

Rotation.

In earlier days, crop rotation was an effective method to control the CRW insect, since root feeding was confined to corn and related grassy weeds. Thus, if corn was not planted, there was no food for the insect. However, as the insect has evolved, it has less fidelity to corn as a crop and also can feed, as the adult, on crops such as soybeans. Thus, the adults lay eggs in soils planted to such crops and even with rotation, root damage by CRW may be significant. Further, the insect has evolved to express an extended diapause. With this variant of the pest, eggs may remain in soil for two years before hatching, thereby avoiding the effects of rotation.

Transgenic resistant varieties.

Corn has been transformed to contain genes encoding three different versions of toxins from Bacillus thuringiensis. These gene products are highly toxic to the insects, but resistance developed almost as soon as the transgenic varieties were introduced. This was in part because high levels of killing require relatively high levels of toxin to be produced, and many varieties had insufficient levels to kill all of the pest population. If a low level of pests survive this first onslaught of the new toxic species, this low level of survivors probably is more resistant to the expressed material. Therefore, resistance to all three of the transgene toxins has developed.

The discussion above strongly indicates that we need new tools to deal with CRW. Applicant has developed microbial tools that significantly improve plant performance. The microbial tools developed provide abilities to endophytically colonize plant roots. These endophytic microbes can be applied as seed treatments or in numerous other ways that allow them to colonize plant roots where they provide systemic beneficial changes in the plant physiology.

The microbes and products based upon such novel microbial compositions are a preventative biological fungicide and nematicide that enhance plant growth and yield and minimize the effects of root pathogens such as Fusarium, Rhizoctonia and Pythium (examples of parasitic fungi), through control or suppression of the pathogens and through root growth enhancement and that provide nematode control plus systemic disease control that provide control from pathogens such as leaf blights. Effects last throughout the season and provide systemic resistance to stresses such as drought, salt and flooding. These materials are compatible with chemical seed treatments making them very amenable to large-scale agricultural practices.

These advantages and related treatment methods have been documented in patents and patent applications including U.S. Pat. No. 8,716,001, PCT/US16/30392 and co-terminally filed provisional patent application No. 62/479,074 entitled Enhanced Microbial and Biorational Control of Nematode Pests of Plants, each of which are incorporated by reference in their entirety.

Demonstration of efficacy.

Seeds of commercial corn variety without Bt traits or fungicides were treated with the following strains identified in Table 1 and sent for laboratory testing by Applicants.

TABLE 1 Treatment # Organism 1 Trichoderma virens strain K1 (ATCC 20906) 2 Trichoderma afroharzianum strain K2 (ATCC PTA 9708) 3 Trichoderma afroharzianum strain K3 (ATCC PTA 9709) 4 Trichoderma atroviride strain K4 (ATCC PTA 9707) 5 Trichoderma atroviride strain K5 (NRRL B-50520) 6 Bacillus spp. TR4 7 Bacillus spp. PH1 Untreated control 9 K2 + K4 (SabrEx corn) 10  K2 + K3 (SabrEx wheat)

The procedure used for the evaluation is as follows:

Procedure for evaluating western corn rootworm larvae infested on corn seeds provided for a trial. The trial was set up to evaluate the ten different seed treatments.

The seeds were planted on 26 Jan. 2017 in 4-inch plastic pots with three seeds per pot. These pots were placed in the green house to get fast germination and growth. On 8 Feb., each pot was thinned to two plants. The next day, each was infested with 25 neonate western corn rootworm non-diapausing larvae, which is the only type of corn rootworm larvae that Applicant was allowed to use in these types of studies by the US Animal and Plant Health Inspection Service.

On 23 Feb. 2017, each plot was evaluated for the number of corn rootworms surviving, the instar of each larvae, the plant height, the plant root length and the weight of each root mass in grams. The length of time from 9 February to 23 February is the approximate time for western corn rootworm larvae to go through three larval instars. At this time, the CRW should be near the prepupal stage of development.

The results are as follows and as indicated in the several FIGS. Data with dissimilar letters are significantly different at P=0.10.

FIG. 1 is a chart showing root damage ratings of corn plants grown with several different microbial treatments. Most of the treatments showed reduced root damage. FIG. 2 is a chart showing plant height of corn plants grown with several different microbial treatments. Many of the treatments increased plant heights in the presence of root damage caused by the CRW. FIG. 3 is a chart showing root weight of corn plants grown with several different microbial treatments. In most of the cases, treated root weights were greater than the untreated control root weights in the presence of CRW.

There were not significant reductions in the number of larvae present on roots as influenced by the microbial agents. This is in contrast with results with nematodes (a very different pest) where not only were root symptoms reduced but numbers of numbers of the pests were reduced.

These data support the following:

Treatment with the disclosed organisms on seeds alleviated damage on roots, even though the number of larvae on roots were not reduced. This is indicative of a mechanism by which tolerance is induced in the corn to CRW. In addition, the microbial organisms used for treatment also increased root and plant growth and, at the same time, reduced damage to roots by pathogens and nematodes.

Microbial treatment methods other than seed treatments are also contemplated by this disclosure. Since the disclosed treatment methods are effective as a seed and as a foliar treatment, there are various other embodiments and applications that are effective, such as, e.g., including: applications as an in-furrow granule; application as a soil drench where the organism will come into contact with roots and colonize roots; application as a root treatment, e.g, during transplant operations; and as a component of liquid or solid fertilizers.

This illustrates that the treatments described herein are a unique tool to limit damage of CRW to non-damaging levels. Not only is damage lessened but the increase in root growth and control of other pests provides a method to limit damage to yields and productivity by CRW and other root attacking pests.

The present disclosure contemplates that many changes and modifications may be made. Therefore, while the presently-preferred form of the system has been shown and described, and several modifications and alternatives discussed, persons skilled in this art will readily appreciate that various additional changes and modifications may be made without departing from the scope of the disclosure, as defined and differentiated by the following claims. 

What is claimed is:
 1. A method for treating plants or plant seeds and increasing plant tolerance to corn rootworms comprising: applying an agent to one or more locations in, on, around, proximal or by a plant, seed, crop, or root, wherein the application occurs at the location and in such a way that impacts the plant or organism of interest in a beneficial manner; wherein the agent includes a microbial metabolite, or mixture of microbial metabolites, that increases the resistance of plants to pests and diseases or otherwise improves plant performance or resistance to stress.
 2. The method of claim 1, wherein the metabolite is derived from Trichoderma spp.
 3. The method of claim 2, wherein the metabolite is selected from the group consisting of Trichoderma virens strain K1 (ATCC 20906), Trichoderma afroharzianum strain K2 (ATCC PTA 9708), Trichoderma afroharzianum strain K3 (ATCC PTA 9709), Trichoderma atroviride strain K4 (ATCC PTA 9707), and Trichoderma atroviride strain K5 (NRRL B-50520).
 4. The method of claim 1, wherein the metabolite is derived from Bacillus spp.
 5. The method of claim 4, wherein the metabolite is selected from the group consisting of Bacillus spp. TR4 and Bacillus spp. PH1.
 6. The method of claim 1, wherein the agent is applied using an in-furrow application method.
 7. The method of claim 1, wherein the agent is applied using a soil drench application method.
 8. The method of claim 1, wherein the agent is used to coat a granule designed for application to agricultural soil or plant medium systems.
 9. The method of claim 8, wherein the granule coated is a plant fertilizer.
 10. The method of claim 1, wherein the result is an enhancement of plant growth.
 11. The method of claim 1, wherein the result is an enhancement of plant biotic stress resistance.
 12. The method of claim 1, wherein the result is an enhancement of plant abiotic stress resistance.
 13. The method of claim 1, wherein the result is an enhancement of corn rootworm resistance.
 14. A composition for treating plant seeds and increasing plant tolerance to corn rootworms comprising a microbial metabolite, or mixture of microbial metabolites.
 15. The composition of claim 14, wherein the metabolite is derived from Trichoderma spp.
 16. The composition of claim 15, wherein the metabolite is selected from the group consisting of Trichoderma virens strain K1 (ATCC 20906), Trichoderma afroharzianum strain K2 (ATCC PTA 9708), Trichoderma afroharzianum strain K3 (ATCC PTA 9709), Trichoderma atroviride strain K4 (ATCC PTA 9707), and Trichoderma atroviride strain K5 (NRRL B-50520).
 17. The composition of claim 14, wherein the metabolite is derived from Bacillus spp.
 18. The composition of claim 17, wherein the metabolite is selected from the group consisting of Bacillus spp. TR4 and Bacillus spp. PH1. 